This invention relates to synthetic processes and intermediates useful for preparing N-arylacridancarboxylic acid derivatives. The N-arylacridancarboxylic acid derivatives are useful in methods to produce chemiluminescence, for example by reaction with a peroxide and a peroxidase. The chemiluminescent reaction is useful in methods of analysis for detecting peroxidase enzymes or hydrogen peroxide. It is also useful in methods to detect and quantify various biological molecules wherein a peroxidase is used as a label and in methods to detect oxidase enzymes which generate hydrogen peroxide.
The detection and quantitation of biological molecules has been accomplished historically with excellent sensitivity by the use of radiolabeled reporter molecules. Recently numerous non-radioactive methods have been developed to avoid the hazards and inconvenience posed by these materials. Methods based on enzyme-linked analytes offer the best sensitivity since the ability to catalytically turn over substrate to produce a detectable change achieves an amplification. Substrates which generate color, fluorescence or chemiluminescence have been developed, the latter achieving the best sensitivity.
Further increases in assay sensitivity will expand the range of utility of chemiluminescence-based methods by permitting the detection of analytes present in smaller quantities or reducing the amount of time and/or reagents required to perform the assay. A way to increase the speed and sensitivity of detection in an enzymatic chemiluminescent assay is through the use of substrates which generate light with a higher efficiency or for a greater length of time.
Among the enzymes used in enzyme-linked detection methods such as immunoassays, detection of oligonucleotides and nucleic acid hybridization techniques, the most extensively used to date has been horseradish peroxidase. Chemiluminescent reagents known in the art do not permit full advantage to be taken of the beneficial properties of this enzyme in analysis mainly due to sensitivity limitations. A reagent which permits the detection of lower amounts of enzyme is needed to enable the use of peroxidase conjugates in applications requiring ultrasensitive detection. Specifically, reagents are required which generate higher levels of chemiluminescence without an accompanying increase in the background or non-specific chemiluminescence. The increased chemiluminescence can be accomplished via either a higher maximum intensity or a longer duration than compounds known in the art.
a. Enzymatic oxidation of N-alkylacridancarboxylic acid derivatives. Applicants"" U.S. Pat. Nos. 5,491,072, 5,523,212, 5,593,845, 5,670,644, 5,723,295 and 5,750,698 disclose the use of a peroxidase enzyme to oxidize substituted and unsubstituted N-alkylacridancarboxylic acid derivatives to generate chemiluminescence. In the presence of a peroxidase enzyme and a peroxide, N-alkylacridancarboxylic acid derivatives are efficiently oxidized to produce the N-alkylacridone and blue chemiluminescence. N-aryl-substituted acridan-carboxylic acid derivatives are not disclosed.
U.S. Pat. No. 6,030,803 discloses a group of acridancarboxylic acid derivatives having a substituted alkoxy or alkylthio leaving group but not aryloxy or arylthio leaving groups as chemiluminescent substrate for peroxidase enzymes. N-aryl acridan compounds are claimed but no examples of N-aryl compounds are provided. All exemplary compounds contain a methyl group as the substituent on the acridan ring nitrogen atom. U.S. Pat. No. 6,162,610 discloses a group of acridancarboxylic acid derivatives having a substituted alkoxy, alkylthio or amide leaving group as chemiluminescent substrate for peroxidase enzymes. The claimed compounds bear a group designated xe2x80x94OX alleged to be a triggering group. No examples of N-aryl compounds are provided nor is a basis for the alleged triggering effect.
N-arylacridancarboxylic acid derivatives having a heteroaromatic group bound to the nitrogen atom are not taught or suggested in this or any of the cited publications nor in any other publication prior to the present invention.
It is therefore an object of the present invention to provide processes for the synthesis of N-arylacridancarboxylic acid derivatives for use in generating chemiluminescence. It is another object of the present invention to provide synthetic intermediates useful in methods for preparing N-arylacridancarboxylic acid derivatives. It is still another object of the present invention to provide N-arylacridancarboxylic acid derivatives for use in generating chemiluminescence. It is also an object of the present invention to provide N-arylacridancarboxylic acid derivatives for use in methods of analysis and detection. It is a further object to provide chemiluminescent methods for the detection of biological materials and compounds. It is also an object of the present invention to provide a chemiluminescent method for detecting peroxidase enzymes and enzyme-conjugates. Additionally, it is an object of the present invention to provide improved methods for use in solution or on surfaces in nucleic acid assays, protein-binding assays, Western blots, Southern blots and other DNA and RNA hybridization assays and for detection of haptens, proteins and antibodies in enzyme immunoassays.
The term xe2x80x9csubstitutedxe2x80x9d when used to describe an organic moiety such as a chain or ring group refers to the replacement of one or more hydrogen atoms on the chain or ring with another atom or group. Exemplary groups include halogen, trihalomethyl, nitro, nitroso, cyano, ammonium, hydrazinyl, carboxyl, carboxamide, carboalkoxy, formyl (xe2x80x94CHO), keto, amino, substituted amino, imino, amido, aryl, alkyl, perfluoroalkyl, alkenyl, alkynyl, alkoxy, hydroxy, sulfhydryl, alkylthio, sulfate, sulfonate, phosphonium, phosphate and phosphonate groups.
The term xe2x80x9cleaving group abilityxe2x80x9d as used herein refers to the propensity for a group when attached to the carbonyl group of the acridancarboxylic acid derivative to be displaced in the nucleophilic reaction of the invention involving a peroxide or hydroperoxide or its anion.
The present invention relates to N-arylacridancarboxylic acid derivatives of the formula: 
wherein R1 is selected from alkyl, substituted alkyl, heteroalkyl, aralkyl, substituted aralkyl, aryl, substituted aryl and heteroaryl groups, R2 to R9 are independently selected from substituents which contain from 1 to 50 atoms selected from C, H, N, O, S, P and halogen atoms, wherein Ar is an aryl, substituted aryl or heteroaryl group and Z is selected from O and S atoms or the group ZR1 is an xe2x80x94NR10R11 group wherein R10 and R11 are independently selected from alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, alkylsulfonyl and arylsulfonyl groups, and wherein R10 and R11 can be combined with N into a heterocycle with leaving group ability including pyrazole, imidazole, benzimidazole, triazole, benzotriazole, tetrazole, oxazole, benzoxazole, thiazole and benzothiazole.
When R10 or R11 is alkylsulfonyl or arylsulfonyl it is preferably selected from methanesulfonyl, trifluoromethanesulfonyl, benzenesulfonyl or substituted benzenesulfonyl and the other of R10 or R11 is preferably an alkyl, phenyl or substituted phenyl group. When R1 is a substituted alkyl or substituted aryl group the group is preferably substituted with one or more electron withdrawing groups, preferably halogen atoms and most preferably with fluorine.
Compounds having Formula I which contain an N-aryl group are useful in methods for producing chemiluminescence. These compounds distinguish from known prior art compounds which all have an alkyl group substituted on the ring nitrogen atom, usually a methyl group. In contrast, the compounds of the present invention bear an aromatic or heteroaromatic ring group on the ring nitrogen. Representative aryl groups include phenyl, naphthyl, biphenyl, anthryl, pyrenyl, pyridyl, quinolyl, acridinyl, furyl, xanthenyl, thienyl, thioxanthyl, thiazolyl, benzothiazolyl, indolyl, imidazolyl and pyrrolyl groups. The aryl or heteroaryl group can be substituted with one or more substituents as defined above and which allows or does not interfere with or prevent the production of light from reaction of the N-arylacridan-carboxylic acid derivative with an oxidant. Representative substituents which can be present on the aryl group include without limitation, halogen, trihalomethyl, nitro, nitroso, cyano, ammonium, hydrazinyl, carboxyl, carboxamide, carboalkoxy, formyl (xe2x80x94CHO), keto, amino, substituted amino, imino, amido, aryl, alkyl, perfluoroalkyl, alkenyl, alkynyl, alkoxy, hydroxy, sulfhydryl, alkylthio, sulfate, sulfonate, phosphonium, phosphate and phosphonate.
Examples of some preferred compounds are: 
wherein X is an electron withdrawing group, Y is a non-hydrogen substituent and wherein m and n are each integers from 0 to 5 and R10 and R11 are as defined above. Preferably R10 is alkylsulfonyl or arylsulfonyl and R11 is alkyl or aryl. Preferred electron withdrawing groups are halogen atoms, more preferably chlorine or fluorine atoms. The number of such electron withdrawing groups m is preferably at least one and more desirably at least two.
Another class of preferred compounds is: 
wherein X, m, R10 and R11 are as defined above and Np is a naphthyl group.
Yet other preferred compounds have the formula: 
wherein alkyl-Xm is a substituted alkyl and X, Y and m and n are as defined above and Z is O or S.
The present invention further relates to novel processes and synthetic intermediates which are used to prepare the N-arylacridancarboxylic acid derivatives. Applicants have discovered new processes for preparing N-arylacridancarboxylic acid derivatives (I) 
beginning from acridone or a ring-substituted acridone compound having the formula: 
Where not commercially available, acridones can be prepared by art-known methods including 1) cyclization of 2-amino-2xe2x80x2-halobenzophenone derivatives, 2) cyclization of diphenylamine-2-carboxylic acids, 3) hydrolysis of 9-chloroacridines and 9-methoxyacridines and 4) rearrangement of 3-phenylanthranils as described in Acridines, R. M. Acheson, ed. Wiley, (1973) Chapter III, pp. 143-196. The acridone compound is reduced to the corresponding acridan by reduction of the ketone moiety. Reduction can be achieved with known reagents for ketone reductions as disclosed In the aforementioned Acridines, pp. 201-2, including Na/Hg amalgam, Al/Hg amalgam, copper chromite, NH2NH2, base and ethylene glycol. In another method the acridone is converted to the 9-chloroacridine compound and then reduced with Raney nickel. In addition, the ketone can be reduced with hydride reducing agents such as LiAlH4.
The substituted or unsubstituted acridan compound thus formed is converted to the N-arylacridancarboxylic acid derivative by a process involving an N-arylation reaction and a reaction process for attaching the carboxylic acid derivative moiety xe2x80x94C(xe2x95x90O)ZR1. The arylation and carboxylate attaching steps can be performed at different points in the synthetic scheme as described in more detail below.
In a first embodiment of the synthetic process, the acridan compound is reacted with an arylating compound selected from aryl halides and sulfonate esters in an inert solvent in the presence of a base and a palladium catalyst to form an N-arylacridan compound. 
In this context aryl includes both aromatic and heteroaromatic ring compounds. Halides include iodide, bromide and chloride. Sulfonate esters include trifluoromethanesulfonates (triflates) and other esters active as leaving groups. Preferred palladium catalysts are prepared from a divalent palladium compound PdL2 and a tertiary phosphine PR3 wherein each R is independently selected from alkyl and aryl groups Suitable divalent compounds PdL2 include any divalent palladium compound with labile ligands selected from carboxylate esters, halogens and ketones and include palladium acetate, palladium chloride, palladium bis(dibenzylideneacetone) Pd(dba)2 and Pd2(dba)3. Suitable tertiary phosphines include trialkylphosphines such as P(t-Bu)3, triaryl phosphines such as BINAP and mixed alkylarylphosphines such as DPPE, DPPF, DPPB and DPPP. Bases include KHPO4, CsCO3, and alkoxide salts such as sodium t-butoxide. Inert solvents useful in this step include toluene, benzene, THF, DME, diglyme and the like. The solvent preferably has a boiling point above about 50xc2x0 C. to enable heating of the reaction. However the reaction can be performed at room temperature or elevated temperatures.
The N-arylacridan compound is then converted to an N-arylacridancarboxylic acid derivative by a carboxylation reaction in which a carbonyl-containing group is attached to the 9-position of the acridan ring. The carbonyl-containing group can be a carboxyl group (xe2x80x94COOH) or its salt, a carboxyl ester group (xe2x80x94COOR1), a thioester group (xe2x80x94COSR1) or an amide group (xe2x80x94CONR10R11). Conversion of the N-arylacridan to the carboxylic acid derivative involves formation of the acridan anion at the 9-position by treatment with a strong base and then reaction with a reagent to attach the carbonyl-containing group.
This conversion can be accomplished in two steps by reaction of the N-arylacridan with a base to generate the anion at the 9-position and capture of the anion with CO2 to produce the N-arylacridancarboxylic acid or its salt. The N-arylacridancarboxylic acid is subsequently converted to any of the various acid derivatives (I) having the group Z-R1 by reaction of the N-arylacridancarboxylic acid with a compound HZ-R1 where Z and R1 are as defined above. Preferably a coupling agent is used to promote the conversion of the acid to the acid derivative. In one embodiment the N-arylacridancarboxylic acid is first converted to the acid chloride by methods generally known in the art such as by use of thionyl chloride (SOCl2) or PCl3. The acid chloride is reacted with a compound of the formula HZ-R1 in the presence of a base or with a salt of the compound HZ-R1. In another embodiment the acid is coupled to the compound HZ-R1 with the aid of a carbodiimide coupling agent such as dicyclohexylcarbodiimide or with carbonyl diimidazole, CDI. In other embodiments strong acids or bases are used as the coupling agent to catalyze the formation of the acid derivative.
Conversion of the N-arylacridan to the N-arylacridancarboxylic acid derivative can also be accomplished in one step by reaction of the 9-position anion described above with a reagent having the formula X-CO-ZR1 which attaches one of the ester (xe2x80x94COOR1), thioester (xe2x80x94COSR1) or amide (xe2x80x94CONR10R11) groups directly. Suitable reagents have a leaving group X such as a halogen attached to the carbonyl group of the carboxylating agent. Suitable reagents would therefore include chloroformate esters (Clxe2x80x94COOR1), chlorothioformate esters (Clxe2x80x94COSR1) and carbamoyl chlorides (Clxe2x80x94CONR10R11).
The present invention further relates, in a second embodiment, to a synthetic process for preparing an N-arylacridancarboxylic acid derivative (I) in which the N-arylation step is performed after formation of the acridancarboxylic acid or acid derivative. In an exemplary process (a) an acridan-9-carboxylic acid derivative or an acridan-9-carboxylic acid is reacted with an arylating agent and a palladium catalyst as described above to effect N-arylation. When the N-arylation reaction is performed on the carboxylic acid, the product N-arylacridancarboxylic acid is then converted to the acid derivative (I) by reaction with a compound HZ-R1 where Z and R1 are as defined above. Preferably a coupling agent as described above is used to promote the conversion of the acid to the acid derivative. 
Acridine-9-carboxylic acid is available commercially and can be prepared using methods known to one of skill in the art of organic chemistry by consultation of the scientific literature. Ring substituted acridan-9-carboxylic acids can similarly be prepared by like methods.
The present invention further relates, in a third embodiment, to a synthetic process for preparing an N-arylacridancarboxylic acid derivative (I) in which the acridone or ring-substituted acridone undergoes the N-arylation step according to the above-described N-arylation reaction method. The N-arylacridone intermediate is then reduced to the corresponding N-arylacridan by reduction of the ketone moiety according to one of the methods described above. The N-arylacridan compound is then converted to an N-arylacridancarboxylic acid derivative by a carboxylation reaction in which a carbonyl-containing group is attached to the 9-position of the acridan ring via either the one step or two step processes as described above 
Another aspect of the present invention relates to synthetic intermediates used in preparing N-arylacridancarboxylic acid derivatives. In particular, N-arylacridancarboxylic acid compounds (II) and their carboxylate salts are claimed wherein R2 to R9 are independently selected from substituents which contain from 1 to 50 atoms selected from C, H, N, O, S, P and halogen atoms, wherein Ar is an aryl, substituted aryl or heteroaryl group. 
Representative aryl groups include phenyl, naphthyl, biphenyl, anthryl, pyrenyl, pyridyl, quinolyl, acridinyl, furyl, xanthenyl, thienyl, thioxanthyl, thiazolyl, benzothiazolyl, indolyl, imidazolyl and pyrrolyl groups. The aryl or heteroaryl group can be substituted with one or more substituents defined as defined above and which allows or does not interfere with or prevent the production of light from the N-arylacridancarboxylic acid derivative when it is reacted with a peroxide and a peroxidase. Preferably, Ar is selected from phenyl, substituted phenyl and naphthyl groups. Representative substituents which can be present on the aryl group include without limitation, halogen, trihalomethyl, nitro, nitroso, cyano, ammonium, hydrazinyl, carboxyl, carboxamide, carboalkoxy, formyl (xe2x80x94CHO), keto, amino, substituted amino, imino, amido, aryl, alkyl, perfluoroalkyl, alkenyl, alkynyl, alkoxy, hydroxy, sulfhydryl, alkylthio, sulfate, sulfonate, phosphonium, phosphate and phosphonate. In preferred compounds of formula (II) each of R2 to R9 are hydrogen or one of R2 to R9 is an alkoxy group and each of the others is hydrogen. When compound (II) is present in the form of a salt, the counter ion will be the same as the counter ion of the strong base used in the carboxylation reaction. Preferred counter ions include alkali metal ions.
Another aspect of the present invention relates to reaction of N-arylacridancarboxylic acid derivatives (I) of the present invention with an oxidant to generate visible chemiluminescence. In one embodiment, reaction of an N-arylacridancarboxylic acid derivative with a base which can remove the proton at the 9-position of the acridan ring, i.e. the proton xcex1 to the carbonyl, in the presence of molecular oxygen in an aprotic solvent produces chemiluminescence. Suitable bases include, hydroxide salts, alkoxide salts such as sodium methoxide and potassium t-butoxide, and tetraalkylammonium fluoride. Aprotic solvents useful include dimethyl sulfoxide, dimethylformamide, dimethylacetamide and tetrahydrofuran.
In another method, reaction of an N-arylacridancarboxylic acid derivative with an oxidant system comprising a peroxide and a peroxidase enzyme produces chemiluminescence. This reaction system is highly useful for assay applications. The chemiluminescence is believed to arise from the excited state of N-arylacridone or the substituted N-arylacridone product as shown in the generalized reaction below. 
Compounds of the present invention typically produce light over a 100-200 nm wide band of emission, which exhibits a maximum intensity at wavelengths in the near ultraviolet to the visible region of the electromagnetic spectrum. Typical wavelengths of maximum intensity xcexmax are in the range of 350-500 nm. It is contemplated that compounds of formula I bearing a covalently linked fluorophore could undergo intramolecular energy transfer resulting in emission at longer wavelengths from the excited state of the fluorophore.
The peroxidase which can undergo the chemiluminescent reaction include lactoperoxidase, microperoxidase, myeloperoxidase, haloperoxidase, e.g. vanadium bromoperoxidase, horseradish peroxidase, fungal peroxidases such as lignin peroxidase and peroxidase from Arthromyces ramosus and Mn-dependent peroxidase produced in white rot fungi, and soybean peroxidase. Other peroxidase mimetic compounds which are not enzymes but possess peroxidase-like activity including iron complexes and Mn-TPPS4 (Y.-X. Ci, et al., Mikrochem. J., 52, 257-62 (1995)) are explicitly considered to be within the scope of the meaning of peroxidase as used herein. Conjugates or complexes of a peroxidase and a biological molecule can also be used in the method for producing chemiluminescence, the only proviso being that the conjugate display peroxidase activity. Biological molecules which can be conjugated to one or more molecules of a peroxidase include DNA, RNA, oligonucleotides, antibodies, antibody fragments, antibody-DNA chimeras, antigens, haptens, proteins, lectins, avidin, streptavidin and biotin. Complexes including or incorporating a peroxidase such as liposomes, micelles, vesicles and polymers which are functionalized for attachment to biological molecules can also be used in the methods of the present invention.
Compounds of the present invention are useful in a reagent composition which generates light in the presence of a peroxidase. Compositions comprise the acridan and a peroxide compound in aqueous solution, preferably a buffer solution, wherein the peroxide participates in the reaction of the acridan with the peroxidase. Optionally the composition can comprise any or all of the following additional components:
a compound which enhances light production from the chemiluminescent reaction;
a chelating agent which prevents the peroxide compound from reacting prior to addition of the peroxidase to the composition; and/or
a surfactant including nonionic, anionic and cationic compounds including monomeric or polymeric compounds.
The peroxide component is any peroxide or alkyl hydroperoxide capable of reacting with the peroxidase. Preferred peroxides include hydrogen peroxide, urea peroxide, and perborate salts.
Suitable buffers include any of the commonly used buffers capable of maintaining a pH in the range of about 6 to about 10 for example, phosphate, borate, carbonate, tris(hydroxymethylamino)methane, glycine, tricine, 2-amino-2-methyl-1-propanol, diethanolamine and the like.
Chemiluminescence enhancing compounds usable include art-known compounds which promote the reactivity of the enzyme. Included among these enhancers are phenolic compounds and aromatic amines known to enhance other peroxidase reactions as described in G. Thorpe, L. Kricka, in Bioluminescence and Chemiluminescence, New Perspectives, J. Scholmerich, et al, Eds., pp. 199-208 (1987), M. Ii, H. Yoshida, Y. Aramaki, H. Masuya, T. Hada, M. Terada, M. Hatanaka, Y. Ichimori, Biochem. Biophys. Res. Comm., 193(2), 540-5 (1993), and in U.S. Pat. Nos. 5,171,668 and 5,206,149 which are incorporated herein by reference. Substituted and unsubstituted arylboronic acid compounds and their ester and anhydride derivatives as disclosed in U.S. Pat. Nos. 5,512,451 and 5,629,168, incorporated herein by reference, are also considered to be within the scope of enhancers useful in the present invention. Yet other enhancer compounds are taught in U.S. Pat. Nos. 5,171,668 and 5,206,149. Also included are phenothiazine and phenoxazine compounds as taught in PCT Publication WO97/39142. Preferred enhancers include but are not limited to: p-phenylphenol, p-iodophenol, p-bromophenol, p-hydroxycinnamic acid, p-imidazolylphenol, acetaminophen, 2,4-dichlorophenol, 2-naphthol and 6-bromo-2-naphthol. Mixtures of more than one enhancer from those classes mentioned above can also be employed. These enhancer compounds are thought to act as co-substrates for the peroxidase and undergo a reversible oxidation.
Chelating agents include cation complexing agents wherein the agent can be selected from the group consisting of chelating agents such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), or ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA) and their salts.
Additives which suppress the generation of chemiluminescence from the reaction of hydrogen peroxide and N-arylacridancarboxylic acid derivatives in the absence of peroxidase enzymes are employed to further improve the utility of the invention. It has also been found that certain surfactants including anionic surfactants such as sodium dodecyl sulfate (SDS), cationic surfactants and nonionic surfactants such as polyoxyethylenated alkylphenols, polyoxyethylenated alcohols, polyoxyethylenated ethers, polyoxyethylenated sorbitol esters and the like improve the sensitivity of detection of the peroxidase enzyme in assays of the present invention by providing a larger chemiluminescence signal and a better signal to background ratio. The improvement occurs through minimizing the background chemiluminescence in the absence of added peroxidase, possibly due to a slowing of the autoxidative decomposition of the acridan derivative. The preferred amounts of the various components of a composition of the present invention are set forth below.
This solution is contacted with the peroxidase enzyme which can either be in solution or adhered to a solid support. Optimum concentrations of reagents must be determined individually for each composition. The concentration of acridan compound and enhancer in particular should be optimized with care for each case in order to produce the maximum enhancement of light emission. The detection reaction can be performed over a range of temperatures including at least the range 20-40xc2x0 C. Detection can be conveniently and advantageously carried out at ambient temperature.
Light emitted by the present method can be detected by any suitable known means such as a luminometer, x-ray film, high speed photographic film, a CCD camera, a scintillation counter, a chemical actinometer or visually. Each detection means has a different spectral sensitivity. The human eye is optimally sensitive to green light, CCD cameras display maximum sensitivity to red light, x-ray films with maximum response to either UV to blue light or green light are available. Choice of the detection device will be governed by the application and considerations of cost, convenience, and whether creation of a permanent record is required.
Another aspect of the present invention is a method for detecting a peroxidase enzyme or an analyte linked to or capable of being linked to a peroxidase enzyme in an assay procedure by a chemiluminescent reaction. The method comprises reacting an acridan of formula I with a peroxide and the peroxidase enzyme to produce chemiluminescence, detecting the amount of chemiluminescence and relating the amount of chemiluminescence to the amount of the analyte or enzyme.
The present invention also relates to a method for detecting hydrogen peroxide in an assay procedure by a chemiluminescent reaction. The method comprises reacting hydrogen peroxide and a peroxidase enzyme with an acridan of formula I to produce chemiluminescence and relating the amount of chemiluminescence to the amount of peroxide.
Further, the invention relates to the use of the method to detect and quantify various biological molecules which are bound to this enzyme by chemical bonds or through physical interactions. Further, the invention relates to the use of the method to detect and quantify various biological molecules which have been or are capable of being bound to peroxidase, for example, by using a biotin-labeled analyte and streptavidin-peroxidase conjugate. Other high affinity binding pairs well known in the art such as fluorescein and anti-fluorescein, digoxigenin and anti-digoxigenin or complementary nucleic acid sequences can also be readily employed as a means of linking a peroxidase enzyme to an analyte for the purpose of practicing this invention. The intensity of the resulting chemiluminescence provides a direct measure of the quantity of labeled organic or biological molecule. For example, the method can be used to detect haptens, antigens and antibodies by the technique of immunoassay, proteins by Western blotting, and DNA and RNA by Southern and Northern blotting, respectively. The method can also be used to detect DNA in DNA sequencing applications.
The method can additionally be used to detect hydrogen peroxide generated by enzymes such as cholesterol oxidase, glucose oxidase, glucose-6-phosphate dehydrogenase, galactose oxidase, galactose-6-phosphate dehydrogenase, and amino acid oxidase. The method can also therefore be used as a means to detect the enzymes mentioned above which generate hydrogen peroxide.
In a further embodiment the methods of the present invention can be used for the detection and measurement of enzyme inhibitors. Inhibitors can act reversibly or irreversibly by denaturing the enzyme, irreversibly binding to the enzyme, or by reversibly binding to the enzyme and competing with substrate. For example, peroxidase inhibitors include cyanide, sulfide and high concentrations of hydrogen peroxide. Further it is recognized that some substances are only inhibitory at certain concentrations and can be only partially inhibitory. In a method of detecting an enzyme inhibitor according to the present invention, a compound of formula (1) is reacted with a peroxidase and a peroxide in the presence and in the absence of the inhibitor and the results are compared to determine the presence or amount of the inhibitor. The effect of the inhibitor can decrease the light intensity, slow the rate of rise of light intensity or cause a delay period before light emission begins or any combination of these effects.